Fluorescent Amino Acid Initiated de novo Cyclic Peptides for the Label‐Free Assessment of Cell Permeability

Abstract The major obstacle in applying peptides to intracellular targets is their low inherent cell permeability. Standard approaches to attach a fluorophore (e. g. FITC, TAMRA) can change the physicochemical properties of the parent peptide and influence their ability to penetrate and localize in cells. We report a label‐free strategy for evaluating the cell permeability of cyclic peptide leads. Fluorescent tryptophan analogues 4‐cyanotryptophan (4CNW) and β‐(1‐azulenyl)‐L‐alanine (AzAla) were incorporated into in vitro translated macrocyclic peptides by initiator reprogramming. We then demonstrate these efficient blue fluorescent emitters are good tools for monitoring peptide penetration into cells.


S1. Synthetic procedures
Mestrenova software. 13 C NMR spectra were recorded in the stated solvents with broadband proton decoupling and an internal deuterium lock. The shift values of resonances are quoted to 1 decimal place unless peaks have similar chemical shifts, in which case 2 decimal places are used. Signals were assigned by the analysis of the chemical shifts, 13 C-1 H HSQC and 13 C-1 H HMBC. Purity was determined by LC-MS and all tested compounds were of >95% purity. LC-MS data were obtained on a Waters ACQUITY (Massachusetts, USA) equipped with QSM, QDa and PDA detectors, sample manager FTN-H, quaternary solvent manager, column manager with ACQUITY UPLC BEH C18 1.7 μm, 2.1 x 50 mm column. Electrospray ionization (ES+ and ES-) and Diode Array spectra were obtained for each characterised compound. The gradient method for LC-MS was 95%-5% 0.1% formic acid (FA) in water/ 0.1 % FA in acetonitrile (MeCN/ACN), over 4 minutes, 0.5 ml/min, 1 μL injection. 4-Cyanotryptophan (4CNW) 3, β-(1-Azulenyl)-L-Alanine (AzAla) 4 were prepared according to previously described protocols. [2] Fmoc-4CNW and Fmoc-AzAla were obtained from 3 and 4 following the literature procedure. [3]

N-Chloroacetyl 4-cyanotryptophan (ClAc-4CNW)
N-(Chloroacetoxy) succinimide (60 mg, 0.31 mmol) in THF (2 mL) was added to a stirring suspension of 4CNW 3 (50 mg, 0.22 mmol) in aqueous Na2CO3 (0.1 M, 4 mL) at rt. The reaction mixture was stirred for 1 h at rt. THF was removed in vacuo and the mixture was acidified to pH 2 by the addition of 1 M aqueous hydrochloric acid. The mixture was extracted with dichloromethane (3 x 30 mL). The combined organic phase was washed with water (10 mL), dried over Na2SO4 and concentrated in vacuo. The residue was purified by reverse-phase column chromatography (acetonitrile with 0.5% formic acid/water with 0.5% formic acid, 0-80%) to give the title compound as a colourless solid (46 mg, 0.15 mmol, 69%). 1
Peptide cyclisation was carried out by incubating the linear peptide (< 1 mg/mL) in aqueous buffer containing ammonium solution (0.25 M, pH 7-8). The reaction mixture was shaken for 1 h, lypohilized and purified by HPLC to give the final cyclic peptide.

S1.5. mRNA template synthesis
The mRNA templates 1 and 2 used in this study were constructed by two rounds of overlapping PCR. The PCR product was purified by phenol-chloroform extraction followed by ethanol precipitation. The purified product was then transcribed overnight using T7 RNA polymerase (Thermo Scientific) following the manufacturer's protocol. The RNA was isolated by isopropanol precipitation and further purified by urea denaturing 8% PAGE gel.
Translation of model peptides P2 was performed using a PURExpress TM Δ (aa, tRNA) in vitro protein synthesis kit (NEB) according to the manufacture's protocol. Translation mixtures were prepared on ice by combining 1.0 µL solution A, 1.5 µL solution B, 0.5 µL tRNA, 0.5 µL mRNA template (Template 2, 10 µM), 0.5 µL amino acid mixture (-Trp, supplemented with 3 or 4), 1 µL water. The translation reaction mixture was incubated at 37 °C for 1 h and analyzed MALDI-TOF mass spectrometry as described above.

S4. Fluorescence visualization of translated peptide
In vitro translation reactions expressing peptides CNW-P1, W-P1 were carried out as described above S3. To 10 µL of translated mixture was added 4X Laemli Sample buffer to terminate translation and the resulting mixture were run on a 15% tricine-SDS-PAGE gel as previously described. [6] In gel fluorescence was imaged in a Chemidoc MP Imaging System (Biorad) using stain free conditions (trans-UV 302 nm excitation).

S5. Cell culture and fluorescence microscopy
Human bone osteosarcoma epithelial cells (U2OS, Crick Cell Services) were cultured in 5% CO2 atmosphere and 37 °C in DMEM (Dulbecco's Modified Eagle's Medium, GIBCO) supplemented with 10% FBS (Fetal Bovine Serum, Sigma Aldrich) and Penicillin/Streptomycin (100 µg/mL, GIBCO). Cells were seeded in an 8 well glass bottom µ-Slide (Ibidi) at a density of 200000 cell/well the day before the experiment. The following day, medium was aspirated and 100 μL of OPTI-MEM (GIBCO) was added in each well. Peptides were dissolved in DMSO and diluted to 250 µM in OPTI-MEM to a final DMSO concentration of 2.5%. 25 μL peptide was added to each well to achieve a final peptide concentration of 50 µM and the cells were incubated at 37˚C with 5% CO2 for 20 or 1440 min (final DMSO concentration 0.5%). After incubation, cells were washed once with OPTI-MEM and imaged in phenol-red free DMEM (GIBCO). Widefield imaging was performed using a Ti Eclipse inverted microscope (Nikon) with motorised XY stage (ASI), using a Plan Fluor 60x/A1.2 WI or Plan Fluor 40x/1.3 NA objective and an Evolve EMCCD camera (Photometrics). The microscope was controlled with Micro-Manager v2.0 gamma software. [7] Fluorescence excitation at 340 nm was performed using a Fura-2 LED light engine (Cairn), a 400 longpass dichroic mirror (T400LP, Chroma) and ET460/50m single bandpass emission filter (Chroma) for 4CNW imaging and a ET395/25X single bandpass emission filter (Chroma) for AzAla imaging. Images were processed with Fiji. [5]

S6. LDH leakage toxicity assay
The protocol was based on an LDH assay previously carried out on peptides. [8] U2OS cells were grown in an identical manner to the microscopy protocol (see S5.). Peptides were added to cells, and after 20 min or 2 h, LDH leakage into cell media was analyzed using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega G1780) according to the manufacturer's protocol. All controls (maximum LDH, vehicle control, cell-free controls) and LDH leakage calculations were conducted as previously reported. [8] Experiments were conducted in triplicate.